Step by Step Guide to Fluorescence Microscopy

The Optical Elements in the Light Path and the Operating Mode of an Inverted Fluorescence Microscope

Fluorescence Microscopy is a special form of light microscopy. It uses fluorescence to highlight structures in fixed and living biological specimens instead of using absorption, phase or interference effects. The fluorescence is delivered either by inorganic dyes, proteins, synthetic beads or by autofluorescent structures within a sample. The most prominent difference between fluorescence microscopy and brightfield-based contrasting methods is that it employs incident light instead of transmitted light. Therefore, the beam path for fluorescence microscopy substantially differs from that of transmitted light techniques.

Nowadays, fluorescence microscopy is regularly used in life science research as it provides the possibility of multicolor staining, the labeling of structures in living cells and even the measurement of the physiological state of a cell. Many present techniques like confocal microscopy and super-resolution microscopy are completely dependent on fluorescence.

In this tutorial the principles of fluorescence microscopy will be explained.

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LeicaMicrosystems FluoScout™

The online tool FluoScout™ helps to determine the optimum filter cube and filter cube set to achieve excellent imaging results. Based on the choice of light source and fluorophores, the user gets a recommendation of filter cubes.